Characterization of Arabidopsis Fluoroacetate-resistant Mutants Reveals the Principal Mechanism of Acetate Activation for Entry into the Glyoxylate Cycle*

The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [14C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle.

[1]  K. Huang.  A sensitive assay method of acetyl CoA synthetase. , 1970, Analytical biochemistry.

[2]  R. Flavell,et al.  Acetate-nonutilizing Mutants of Neurospora crassa I. Mutant Isolation, Complementation Studies, and Linkage Relationships , 1968, Journal of bacteriology.

[3]  Steven Penfield,et al.  Reserve Mobilization in the Arabidopsis Endosperm Fuels Hypocotyl Elongation in the Dark, Is Independent of Abscisic Acid, and Requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1 , 2004, The Plant Cell Online.

[4]  J. Wintermans,et al.  Spectrophotometric characteristics of chlorophylls a and b and their phenophytins in ethanol , 1965 .

[5]  Joseph R. Ecker,et al.  CTR1, a negative regulator of the ethylene response pathway in arabidopsis, encodes a member of the Raf family of protein kinases , 1993, Cell.

[6]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[7]  P. Eastmond,et al.  Re-examining the role of the glyoxylate cycle in oilseeds. , 2001, Trends in plant science.

[8]  J. David,et al.  Molecular evolution of the AMP-forming Acetyl-CoA synthetase. , 2001, Gene.

[9]  J. Ohlrogge,et al.  Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue. , 2000, The Plant journal : for cell and molecular biology.

[10]  R. R. Theimer,et al.  Derepression of glyoxylate cycle enzyme activities in anise suspension culture cells , 1983 .

[11]  D. Walker.  [18] Chloroplasts (and Grana): Aqueous (including high carbon fixation ability) , 1971 .

[12]  J. Bonner,et al.  Acetate activation and acetoacetate formation in plant systems. , 1954, Archives of biochemistry and biophysics.

[13]  G. Turner,et al.  Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method , 1997, Archives of Microbiology.

[14]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[15]  R. Mache,et al.  Arabidopsis A BOUT DE SOUFFLE, Which Is Homologous with Mammalian Carnitine Acyl Carrier, Is Required for Postembryonic Growth in the Light Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002485. , 2002, The Plant Cell Online.

[16]  D. Kuhn,et al.  Subcellular localization of acetyl-CoA synthetase in leaf protoplasts of Spinacia oleracea. , 1981, Archives of biochemistry and biophysics.

[17]  T. K. Lee,et al.  Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose , 1996, Plant physiology.

[18]  J. Sheen,et al.  Metabolic repression of transcription in higher plants. , 1990, The Plant cell.

[19]  Role of Light-harvesting Complex 2 Dissociation in Protecting the Photosystem 2 Reaction Centres Against Photodamage in Soybean Leaves and Thylakoids , 2003, Photosynthetica.

[20]  N. Tolbert.  Isolation of subcellular organelles of metabolism on isopycnic sucrose gradients. , 1974, Methods in enzymology.

[21]  Steven M. L. Smith.  Does the glyoxylate cycle have an anaplerotic function in plants? , 2002, Trends in plant science.

[22]  E. Heinz,et al.  The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal -map and is a new member of the AMP-binding protein family , 1994, Molecular and General Genetics MGG.

[23]  I. Minkov,et al.  Protochlorophyllide and Chlorophyllide in Reformed Prolamellar Bodies and Thylakoids of Irradiated Dark-grown Wheat (Trificum aestivum L.) , 1993 .

[24]  N. Mitsukawa,et al.  Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. , 1995, The Plant journal : for cell and molecular biology.

[25]  M. Hooks.  Molecular Biology, Enzymology, and Physiology of β-Oxidation , 2002 .

[26]  Eve Syrkin Wurtele,et al.  Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis1,212 , 2002, Plant Physiology.

[27]  D. T. Canvin,et al.  Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway. , 1961, The Journal of biological chemistry.

[28]  M. Hayashi,et al.  Novel glyoxysomal protein kinase, GPK1, identified by proteomic analysis of glyoxysomes in etiolated cotyledons of Arabidopsis thaliana. , 2003, Plant & cell physiology.

[29]  Robert Kay,et al.  Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes , 1987, Science.

[30]  Su-il Kim,et al.  Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice , 2003, Biotechnology Letters.

[31]  J. F. Hogg,et al.  Chain length specificities of peroxisomal and mitochondrial beta-oxidation in rat liver. , 1979, Biochemical and biophysical research communications.

[32]  M. A. Hooks,et al.  Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings , 2004, Molecular Genetics and Genomics.

[33]  J. Browse,et al.  Arabidopsis Contains a Large Superfamily of Acyl-Activating Enzymes. Phylogenetic and Biochemical Analysis Reveals a New Class of Acyl-Coenzyme A Synthetases1 , 2003, Plant Physiology.

[34]  V. Germain,et al.  Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  C. Bradbeer,et al.  Fat metabolism in higher plants. XI. The conversion of fat into carbohydrate in peanut and sunflower seedlings. , 1959, The Journal of biological chemistry.

[36]  M. Davis,et al.  The acetate regulatory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional activator , 1997, Molecular and General Genetics MGG.

[37]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[38]  G. Turner,et al.  The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. , 1990, The EMBO journal.

[39]  D. Apirion.  The two-way selection of mutants and revertants in respect of acetate utilization and resistance to fluoro-acetate in Aspergillus nidulans. , 1965, Genetical research.

[40]  R. Dixon,et al.  Activation tagging in Arabidopsis. , 2000, Plant physiology.

[41]  J. Ohlrogge,et al.  Acetyl coenzyme A concentrations in plant tissues. , 2004, Journal of plant physiology.

[42]  I. Graham,et al.  Induction of Malate Synthase Gene Expression in Senescent and Detached Organs of Cucumber. , 1992, The Plant cell.

[43]  G. Turner,et al.  The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine/acylcarnitine carrier , 1999, Archives of Microbiology.